The fatty acid synthesis pathway has gained notoriety as a target for treatment of obesity, certain cancers, and bacterial infections. The mammalian, cytoplasmic fatty acid synthase (FAS) produces the 16-carbon fatty acid, palmitate, through a series of acyl chain elongation and saturation reactions. This multi-enzyme protein must exhibit extensive conformational flexibility to permit transfer of the growing acyl chain between catalytic domains. The recent partial crystal structure of FAS has permitted knowledge of the locations of most of the domains in an earlier cryo-EM structure published by the Asturias and Smith groups (Asturias, 2005);however these structures provide only a static picture of a dynamic enzyme. The goal of this work is to understand the domain motions of FAS during catalysis. We have begun evaluating the conformational variability of FAS by using single particle electron microscopy to visualize mutants that are inactivated in a single catalytic domain and thereby arrested at that catalytic step in the presence of substrates. Our initial work compares the conformations elicited by mutations that arrest catalysis at two different steps. One of these, the C161Q mutation, cannot catalyze the elongation of the acyl chain and appears to have several unique conformations relative to the A22 mutation that slows the release of the completed acyl chain. To quantify the observed conformational changes we have adapted an existing software for a novel purpose to track apparent domain motions in 2D images and will use this method to compare these and other mutants. Using these techniques we will: (1) extend this comparison of conformational variations to mutants that are defective in other catalytic steps, (2) use a cross-linker to specifically capture functional interactions between two domains of FAS and study the conformational flexibility of these cross-linked structures, (3) produce 3D structures of the FAS mutants with substrates and of the cross-linked FAS using standard reconstruction techniques, and (4) use an atomic model of FAS to interpret the flexibility and conformational changes that were observed in 2D and 3D. Using these methods we hope to better understand domain motions of FAS that mediate catalysis. The methods that we use and develop here are likely to be useful for analyzing the conformational flexibility of other macromolecular complexes.